Global regulation by proteolysis in Escherichia coli: Molecular recognition, signal integration and quantitative analysis of proteolysis-controlled regulatory circuits

某些关键调节因子的细胞量是合成和降解调节之间高度动态和调节平衡的结果。以大肠杆菌中核糖核酸聚合酶Sigma-S亚单位的多重胁迫抑制蛋白降解为模型系统，提出了调控蛋白降解领域中两个尚未解决的主要问题：(1)蛋白质分解机制选择性底物识别的分子细节；(2)连接环境胁迫条件和关键细胞调节因子蛋白分解速率的信号转导途径。Sigma-S直接被一个识别因子RSSB靶向，它对Sigma-S(A2.5)内一个小区域的亲和力受磷酸化控制。然后RSSB将Sigma-S转移到ClpXP蛋白酶，在那里Sigma-S被迅速且完全降解。我们将研究Sigma-S(可能在Sigma-70中)的蛋白水解性识别位点与各种识别因子和伴侣(RSSB、ClpX、ClpA)之间的分子相互作用。此外，还将阐明抑制Sigma-S在不同胁迫条件下周转的信号转导途径。这些途径分别控制RSSB的磷酸化和/或Sigma-S和ClpXP的竞争结合伙伴的细胞量和与之的相互作用，例如核心RNA聚合酶或替代的ClpXP底物。这些研究应该会为调控的蛋白分解以及复杂的细菌信号整合网络提供重要的新见解。

The cellular amount of certain key regulatory factors is the result of a highly dynamic and regulated balance between regulation of synthesis and degradation. Using the multiple stress-inhibited proteolysis of the Sigma-S subunit of RNA polymerase in E.coli as a model system, the proposal submitted here focusses on the two major unsolved problems in the field of regulated proteolysis: (i) the molecular details of selective substrate recognition by the proteolytic machinery, and (ii) the signal transduction pathways that connect environmental stress conditions to the rate of proteolysis of a key cellular regulator. Sigma-S is directly targeted by a recognition factor, RssB, whose affinity for a small region within Sigma-S (a2.5) is controlled by phosphorylation. RssB then transfers Sigma-S to the ClpXP protease, where Sigma-S is rapidly and completely degraded. Molecular interactions between proteolytic recognition sites in Sigma-S (and probably in Sigma-70) and various recognizing factors and chaperones (RssB, ClpX, ClpA) will be studied. In addition, the signal transduction pathways that inhibit Sigma-S turnover in response to various stress conditions will be elucidated. These pathways either control RssB phosphorylation and/or the cellular amount of and interaction with competing binding partners for Sigma-S and ClpXP, such as core RNA polymerase or alternative ClpXP substrates, respectively. These studies should provide important novel insights into regulated proteolysis as well as into complex bacterial signal integration networks in general.